American society has become increasingly ‘obesogenic’ via influences of environments that promote increased food intake and physical inactivity. An excessive amount of body fat or adipose tissue contributes to obesity. Adipose tissue is an important endocrine regulator of energy homeostasis and glucose metabolism. New adipocytes are required for storage of excess energy (intake>expenditure) in white adipose tissue (WAT). Excess adipose tissue mass is the basis of obesity and its associated diseases. Adipose tissue mass is determined by adipocyte size and number. Under circumstances of positive energy balance, there is adipose mass expansion (hypertrophia) and hyperplasia.
The cellular components of adipose tissue also include preadipocytes and stem cells residing in adipose stromal-vascular compartments that differentiate to adipocytes. Given proper environmental and hormonal cues, pre-adipocytes undergo clonal expansion and subsequent terminal differentiation into mature adipocytes. During adipogenesis, expression and activity of PPARγ and C/EBP family and their co-factors promote the morphological and functional changes of a primitive, multipotent state to an adipocyte phenotype characterized by cell shape and lipid accumulations (1-3). 3T3L1 murine preadipocyte cell line (4) is widely used as it authentically reproduces adipogenesis including expression of adipogenic genes and morphological changes. Once these cells are terminally differentiated, they undergo growth arrest and form large spherical intracellular lipid droplets. When these cells are implanted into mice, they are histologically indistinguishable from WAT (5, 6).
Preadipocytes undergo apoptosis while mature adipocytes are not susceptible to apoptosis. This was demonstrated in 3T3L1 preadipocytes which go through apoptosis as shown by DNA fragmentation, Hoescht staining and TUNEL (7, 8). Concomitantly, Bcl2 levels increased as the adipose cells differentiated into mature adipocytes (9-11). This suggested a change in gene expression patterns from preadipocytes to mature adipocytes during adipogenesis. An important mechanism of regulating gene expression during differentiation is alternative splicing which expands the coding capacity of a single gene to produce different proteins with distinct functions (12). Many genes in the apoptosis pathway are alternatively spliced. Divergence observed in gene expression due to alternative splicing may be tissue-specific (13, 14), developmentally regulated (15, 16) or hormonally regulated (17, 18).
Protein Kinase C delta (PKCδ) is a serine/threonine kinase which plays a central role in apoptosis. PKCδ has dual effects: as a mediator of apoptosis and as an anti-apoptosis effecter. Its splice variants, PKCδI and PKCδII, are a switch that determines cell survival and fate. PKCδI promotes apoptosis while PKCδII promotes survival (19). PKCδII is the mouse homolog of human PKCδVIII (20). Both are generated by alternative 5′ splice site usage, and their transcripts share >94% sequence homology. It has been shown that PKCδII and PKCδVIII function as pro-survival proteins (21); the functions of the other PKCδ splice variants are not yet established. PKCδII is generated by utilization of an alternative downstream 5′ splice site of PKCδ pre-mRNA exon 9. PKCδII, which is resistant to cleavage by caspase-3, arises from insertion of 78 base pairs (bp) (26 amino acids) in its caspase-3 recognition sequence (DILD) (22). Previously, it was also shown that over expression of PKCδII decreased apoptosis and promoted survival in neuronal cells (19).